Project Summary The overall goal of this proposal is to discover agonist scaffolds for the orphan G protein-coupled receptor 6 (GPR6). GPR6 is selectively expressed in dopamine D2 receptor (D2R) containing neurons of the striatopallidal pathway, which play a critical role in the development and maintenance of drug addiction. GPR6 is constitutively active and signals through G?s to stimulate cAMP production in neurons. GPR6 agonists have the potential to activate the striatopallidal pathway in the nucleus accumbens; thereby, opposing the stimulant and rewarding properties of addictive drugs. GPR6 is an innovative new target for therapeutic intervention in substance use disorders. Currently there are no small molecule GPR6 agonists reported in the literature; therefore, there remains an unmet need for potent, selective GPR6 agonists to help examine receptor signaling, and facilitate drug discovery targeting this receptor. Under this application we will work to discover drug-like agonist probes for GPR6. With this goal in mind, in Aim 1 we will conduct a high throughput screening assay using a stable HEK hGPR6 cell line. We will screen approximately 40,000 compounds from an RTI library designed for scaffold diversity and targeting GPCR pharmacophores. In confirmatory studies, we will determine the potency of select hits in the cAMP assay, counter screen in the parental cell line, and confirm GPR6 specific agonist activity of hits, by inhibition with a GPR6 antagonist. In Aim 2 we will conduct a preliminary structure-activity relationship study. The SAR campaign will be an iterative process between compound purchase/synthesis, in vitro functional characterization of compound activity, and computational/molecular modelling. Lead compounds will also be tested in an orthogonal, secondary functional assay for on-target validation. Upon completion of this grant, we expect to have at least three lead agonist scaffolds identified that are ready for further probe development. Novel GPR6 agonist probes identified under this application will serve as tools to investigate the signaling mechanisms and in vivo functions of GPR6.